Embodiments disclosed herein relate to methods for purification of proteins, including antibodies, including IgG antibodies.
The technique of protein precipitation with non-ionic organic polymers, such as polyethylene glycol (PEG), is known. It is most often used at low salt concentrations, in the absence of added salt, but exceptions are known (D. Gervais et al, US Patent Application 2010/0204455 A1; K. Ingham, Arch. Biochem. Biophys. 186 (1978) 106-113; K. Yamamoto et al, Virology, 40 (1970) 734-744; S. Branston et al, Biotechnol. Progr. 28 (2012) 129-136).
The technique of Steric Exclusion Chromatography is known (J. Lee et al, J. Chromatogr. A 1270 (2012) 162-170; see also PCT/SG2012/000199, incorporated herein by reference and attached as Appendix A). It exploits nonionic hydrophilic surfaces on which retention of a desired product is induced by a nonionic organic polymer such as PEG. Elevated NaCl concentrations have been reported to increase virus binding efficiency (Lee et al supra). Conducting the technique on fluidized particles in the presence of elevated concentrations of NaCl has been reported to increase recovery and purity of IgG preparations (P. Gagnon et al, J. Chromatogr. A, 1324:171-180, (2014).
Methods for reducing the content of chromatin from cell culture harvests containing monoclonal antibodies have been described (H. Gan et al, J. Chromatogr. A, 1291 (2013) 33-40. Chromatin is known to include DNA and histone proteins in stable associations known as nucleosomes. Gan et al reported that chromatin and its catabolites also form associations with antibodies that limit the efficacy of subsequent fractionation methods. They reported methods that achieved chromatin reduction of 99%, and further reported that such reduction improved the quality of purification obtained by cation exchange chromatography. A variation of a technique reported by Gan et al has been reported by Gagnon et al (2013, supra) to improve recovery and purity of IgG fractionated by steric exclusion chromatography on fluidized hydrophilic particles.